Xeroderma pigmentosum (XP) syndrome is a closely related group of autosomal recessive diseases impaired in nucleotide excision DNA repair, manifested as an extreme sensitivity to sunlight (UV) resulting in a very high incidence of skin cancer and frequent neurological abnormalities. The objective of the proposed research study is to molecularly clone and characterize the DNA repair gene(s) isolated from phenotypicly complementated xeroderma pigmentosum group C (XP-C) cells, and to determine whether this, or one of these genes, is defective in XP-C patients. The experimental approach is as follows: 1) Rescue by polymerase chain reaction and molecular cloning of the cDNA(s) responsible for the full restoration of DNA repair proficiency in previously obtained primary XP-C transfectants. 2) Transfection of the rescued cDNA(s) into other XP genetic groups to assess the specificity of complementation. 3) Structural analysis of the complementing cDNA(s). 4) Scanning of genomic DNA and RNA derived from XP-C cells for mutations that inactivate the cloned complementary gene(s). This will be accomplished using techniques such as Southern and Northern blot analysis, ribonuclease A mismatched cleavage, Cotton's chemical cleavage, primer extension with mobility shifting nucleotide analogs and DNA sequencing. 5) Identification and isolation of the protein encoded by the DNA repair gene. 6) Analysis of transcriptional regulation and regulatory cis-elements in the repair gene. Isolation of the XP-C DNA repair gene would open up the possibility of studying the regulation of this essential gene product (is it subject to an SOS-like response?), and the biochemical function(s) of the encoded repair protein. It will also permit the development of prenatal diagnosis, at the DNA level, for XP-C suspected fetuses.
| Dotan, I; Kopatz, I; Naiman, T et al. (1995) Establishment of an autocatalytic conditional mammalian system for expression of stringently regulated genes. Nucleic Acids Res 23:307-9 |
